The Effect of Thyroid Hormone, Prostaglandin E2, and Calcium Gluconate on Orthodontic Tooth Movement and Root Resorption in Rats.

STATEMENT OF THE PROBLEM
A major objective of investigators is to clarify the role of metabolites in achievement of maximum tooth movement with minimal root damage during orthodontic tooth movement (OTM).


PURPOSE
The aim of this study was to determine the effect of administration of thyroid hormone, prostaglandin E2, and calcium on orthodontic tooth movement and root resorption in rats.


MATERIALS AND METHOD
Sixty four male Wistar rats were randomly divided into 8 groups of eight rats each: 1- 20µg/kg thyroxine was injected in traperitoneally after installation of the orthodontic appliance.  2- 0.1 ml of 1 mg/ml prostaglandin E2 was injected submucosally.  3- 10% (200 mg/kg) calcium gluconate was injected.  4- Prostaglandin E2 was injected submucosally and 10% calcium was injected intraperitoneally.  5- Thyroxine was injected intraperitoneally and prostaglandin E2 was injected submucosally.  6- 20µg/kg thyroxine with calcium was injected. 7- Prostaglandin E2 was injected submucosally with calcium and thyroxine.  8- Distilled water was used in control group. The orthodontic appliances comprised of a NiTi closed coil were posteriorly connected to the right first molar and anteriorly to the upper right incisor. OTM was measured with a feeler gauge. The mid-mesial root of the first molar and the adjacent tissues were histologically evaluated. The Data were analyzed by one-way ANOVA and Student-Newman-Keuls test.


RESULTS
The highest mean OTM was observed in the thyroxine and prostaglandin E2 group (Mean±SD = 0.7375±0.1359 mm) that was significantly different (p< 0.05). A significant difference (p< 0.05) in root resorption was observed between the prostaglandin E2 (0.0192±0.0198 mm(2)) and the other groups.


CONCLUSION
It seems that the combination of thyroxine and prostaglandin E2, with a synergistic effect, would decrease the root resorption and increase the rate of orthodontic tooth movement in rats.


Introduction
Remodeling of the surrounding alveolar bone and cellular changes in the periodontal ligament (PDL) underlie orthodontic tooth movement. [1] Reducing the length of treatment may thus help satisfy patients' demands and even lessen the long term sequel. [2] Increasing me-chanical force to reduce the treatment time, a major problem in orthodontic practice, leads to several sequel.
A common major complication of orthodontic treatment is apical root resorption. [3][4][5] Therefore maximum tooth movement with minimal root damage has been a major objective of investigators. [6] One of the factors that are being investigated for their effects on tooth movement is prostaglandins (PGs). [7][8][9] PGs, especially PGE 2 , are potent multifunctional regulators of bone metabolism. [10] PGE 2 induces morphologic changes in osteoclasts and osteoblasts via increased intracellular levels of CAMP (cyclic adenosine monophosphate); [11][12] exogenous PGE 2 increases the mRNA (Messenger RNA) synthesis and protein secretion of the Receptor Activator of Nuclear factor kappa-β Ligand (RANKL). [13] However, there was an increase in the amount of root resorption with increasing the numbers and concentrations of PGE 2 injections. [14][15][16] In addition, various factors influence the amount of root resorption, including the proven effect of systemic calcium. [17] Low levels of calcium causes secondary hyperparathyroidism and induces an increase in the secretion of parathyroid hormone (PTH) as well as vitamin D active metabolites. [18][19][20] In bone, PTH can induce a rapid release of calcium, but it also mediates long-term changes by acting directly on osteoblasts and indirectly on osteoclasts. [21][22] In osteoblasts, PTH affects cellular metabolic activity, gene transcriptional activity, and multiple protease secretion. [23][24][25] Its effects on osteoclasts occur through producing RANKL, a protein that plays a crucial role in osteoclast formation and activity. [26] Thus, the increase of bone turnover induced by PTH could accelerate orthodontic movement and root resorption. [27][28] In addition to parathyroid hormone, bone resorption activity is also regulated by lthyroxine. [29] Thyroid hormone plays a crucial role in normal growth and development of vertebrate bones. [30][31] Administration of high doses of l-thyroxine in rats has been found to increase bone resorption. [32] Thyroid hormones increase osteoclastic bone resorption in rats by stimulating the prostaglandin. [33] Its administration in rats increases the speed of tooth movement.
[34] Application of low doses of thyroid hormones may have a protective effect on the root surface and reduce the extent of root resorption. [35][36][37] The studies performed by Poumpros et al. [36] and Shirazi et al. [35] showed that thyroid hormone administration in rats not only increased the speed of tooth movement, but also reduced the extent of root resorption.
Goldie and King [19] found that systemic calcium deficiency increased OTM. In the study enrolled by Yamasaki et al., [16,18] Kohoe et al., [17] and Boekenoogen et al. [15] that evaluated the rate of OTM and root resorption after injection of PGE2, revealed that increase in osteoclastic activity resulted in increased rate of OTM and root resorption.
So far, no research has been undertaken on injection of thyroxine with PGE or calcium during orthodontic treatment and its effect on root resorption or tooth movement.
The aim of the present study is to compare and investigate the synergistic effect of thyroxine with PGE2, and calcium gluconate on orthodontic tooth movement and root resorption in rats.

Animals
All animal handling and surgical procedures were ap- Sixty four male Wistar rats (6-8 weeks old, 230-300 grams weight) were randomly divided into 8 groups (n=8). They were fed on NIH-36 diet for mice and rats, with a minimum of 1.15 per cent calcium content.
Fresh drinking water was provided every day and they were cared for according to the animal welfare regulations.

Appliance design
The orthodontic appliance comprised of a 5 mm long  The injections were administered on the days 0 and 7.

Histologic evaluation
The animals were sacrificed after 21 days using vapor-  Table 1 illustrates the values obtained regarding OTM in the eight groups with an orthodontic appliance. As the F-test in ANOVA demonstrated a significant difference among the eight groups, a Student's t-test was used   Table 2 illustrates the values obtained for root resorption in the eight groups studied and Figure 2 represents the histological section belonging to the root of a sample from each of the eight groups. Since there was a variance difference in the eight groups, with a p-value close to 0.05, and the data did not follow a normal distribution curve, a Kruskal-Wallis test was used to confirm the presence of a significant difference in root resorption among the groups. Multiple range tests were then used to compare groups in pairs, which revealed a significant difference between the PGE2 and T, T+Ca, T+Ca+ PGE2 groups. No significant difference was observed between the other groups regarding root resorption.

Discussion
In this study, OTM occurred significantly faster in T group compared with the control group (p< 0.01) which was in agreement with the findings of Shirazi et al. [35] The reason for the increase might be the bone-resorptive effect of thyroxine. [30] It directly impacts the bone remodeling, [38] accelerates the osteoclasts activity in rats by stimulating the prostaglandin. In the present study, OTM occurred significantly activity. [36][37][38] Faster in the PGE2 group compared with the control group, which was in line with the findings of Yamasaki et al., [16,18] Kohoe et al., [17] and Boekenoogen et al. [15] This increase might be due to the bone-resorptive effects of PGs after orthodontic loading.
Following periodontal injury due to loading, PG is synthesized and PGE2 increases the mRNA synthesis and protein secretion of the receptor activator of nuclear factor kappa-B ligand (RANKL), [13][14] then osteoclastic activity commences, which leads to bone resorption and tooth movement. [16] Thus adding PGE to a live environment may induce bone resorption. [18] Moreover, OTM occurred slightly lower in the Ca group compared with the control group, which was in accordance with what was found by Goldie and King [19] who observed that systemic calcium deficiency increased OTM. So, the hypoparathyroidism caused by calcium injection in the present study, must have inhibited bone remodeling and resisted the tooth movement.
Combined injection of PGE2 and Ca reduced OTM compared with PGE2 per se; however, despite this decrease it still occurred at a significantly higher rate compared with the control group.
The present results showed that the highest amount of OTM occurred in T+PGE2 group that was significantly higher than T and PGE2 groups. No information was available regarding the combination injection of T+PGE2 during OTM. As mentioned before, thyroid hormones increase osteoclastic bone resorption in rats by stimulating the prostaglandin, [31][32][33][34][35][36][37][38][39] and exogenous PGE2 increases the mRNA synthesis and protein secretion of the receptor activator of nuclear factor kappa-B ligand (RANKL) [13][14] in osteoblasts.
Thus, it can be concluded the bone-resorptive of thyroxine may happen because of RANKL via PGE2.  [1,22] in addition to the root protective effect of thyroxine. [35][36][37][38][39] But since no significant difference was found between the T and T+Ca groups, it can be concluded that addition of Ca to T in order to achieve the minimum root resorption cannot recommended.

Conclusion
The results of the present study indicate that in order to achieve a decrease in root resorption and an increase in

Conflicting Interest
The authors declare no conflict of interest.